PROJECT SUMMARY/ABSTRACT T cells survey the body for potential threats, including tumor cells, by probing their environment through physical interactions. During these physical interactions, molecular forces generated by the T cell receptor (TCR) ?pulling? on potential threats help discriminate antigenic tissue from self-tissue by strengthening and prolonging TCR bonds, thus causing an enhanced T cell activation signal. This is an intriguing concept, as a force pulling on a molecular bond would be thought to cause rapid bond dissociation and loss of signaling. As this mechanical force is critical for T cell activation and function, the role of molecular forces within the immune system should be explored in other TCR co-receptor interactions as well. This includes the interaction between programmed death receptor 1 (PD1) on T cells and programmed cell death ligand 1/2 (PDL1/2) on tumor cells; an interaction that is revolutionizing cancer treatment but is currently still poorly understood. Specifically, it has been observed that when an active T cell physically crawls on and engages a tumor cell expressing PDL1 or PDL2, the T cell shuts down and loses its effector functions due to PD1 signaling, but through an unknown mechanism. As PD1 closely associates with the TCR, we hypothesize that the PD1 receptor is also force-regulated, and that molecular-level mechanical forces are also critical to PD1 signaling. The goal of our research is to determine the mechanistic role of mechanical forces in PD1 signaling, which we will accomplish through the following three aims. Aim 1. Create and characterize recombinant PDL1/PDL2 force sensors to quantify PD1 forces in cytotoxic T cells. Using molecular probes previously developed in our lab, we will design PDL1/2 force sensors that consist of a hybridized DNA duplex functionalized with a fluorophore-quencher pair. Upon force application by the PD1 receptor, the DNA duplex is mechanically unfolded, causing separation of the fluorophore and quencher. This increase in fluorescence can be mapped and quantified using conventional fluorescence microscopy, as the DNA duplex can be tuned to display specific forces exerted by the receptors. The results of this study will allow us to obtain the first maps of PD1 molecular forces in T cells. Aim 2. Determine the amino acids that contribute to the mechanical stability of the PD1-PDL1/2 complex. We will use site-directed mutagenesis to generate a mutant library of PDL1 and PDL2 to determine which amino acids contribute to molecular forces. Aim 3. Determine the role of PD1 molecular forces in T cell signaling. We will mechanically cap the forces the PD1 receptor can transmit and relate this information to intracellular signaling. The results of these studies will establish the role of PD1 mechanics in T cell signaling and will provide insight to the molecular events occurring during the physical ?kiss of death.? Understanding the paradigms of mechanical forces in T cell biology and tumor recognition will greatly expand our knowledge of tumor-immune system interactions and will open doors to new avenues of cancer immunotherapies.